The anxiety of withdrawal from chronic ethanol exposure has been implicated in sustaining alcohol abuse. Work in our laboratory during the previous granting period demonstrated that repeated withdrawal from chronic ethanol exposure led to a sensitization of anxiety that accompanied withdrawal. Serotonin.a major neurotransmitter in brain.and the 5-HT2C and 5-HT1A receptor subtypes were implicated in this sensitization process associated with repeated withdrawals. The purpose of the present series of nvestigations is to extend these data to test specific hypotheses that define specific brain regions where the 5-HT2C and 5-HT1A receptors contribute to the sensitization of anxiety and that account for the neurobiological basis of the sensitization of the anxiety. Specific Aim 1 will test the hypothesis that release of serotonin in the amygdala is responsible for the sensitization of anxiety induced by multiple withdrawals from chronic ethanol exposures. This work will include microinjection studies to establish the importance of 5-HT2C receptors at this site in the withdrawal-induced anxiety. Additionally, studies will explore whether the raphe has a role in the release of serotonin during withdrawal from the multiple ethanol exposures that results in a sensitization of anxiety. Specific Aim 2 will test the hypothesis that 5-HT2C and 5-HT1A receptor number or sensitivity will be enhanced in selected brain regions by repeated withdrawals as contributing factors in the sensitization of anxiety following repeated withdrawals. Specific Aim 3 of this proposal will test the hypothesis that specific proteins in the second messenger cascade down-stream from the 5-HT2C and 5-HT1A receptors are persistently altered and are critical for sustaining the persistent anxiety induced by repeated withdrawals. Emphasis will be on the G-protein coupled pathways linked to PLC and adenylyl cyclase. Complementing these biochemical measures will be the use of "knock outs" of specific genes associated with proteins in the 5-HT2C and 5-HT1A receptor second messenger cascades to test in vivo the importance of specific messenger proteins in the pathways in sustaining the sensitization of anxiety observed with repeated withdrawals from chronic ethanol.